Project description:The transcription factor HY5 acts downstream of multiple families of the photoreceptors and promotes photomorphogenesis. Although it is well accepted that HY5 acts to regulate target gene expression, in vivo binding of HY5 to any of its target gene promoters has yet to be demonstrated. Here we used a chromatin immunoprecipitation procedure to verify suspected in vivo HY5 binding sites. We demonstrated that in vivo association of HY5 with promoter targets is not altered under distinct light qualities or during light-to-dark transition. Coupled with DNA chip hybridization using high density 60-nucleotide oligomer microarray that contains one probe for every 500 nucleotides over the entire Arabidopsis genome, we mapped genome wide in vivo HY5 binding sites. This analysis showed that HY5 binds preferentially to promoter regions in vivo and revealed over 3 thousand chromosomal sites as putative HY5 binding targets. HY5 binding targets tend to be enriched in the early light responsive genes and transcription factor genes. Our data thus supports a model in which HY5 is a high hierarchical regulator of the transcriptional cascades for photomorphogenesis. Keywords: ChIP-chip
Project description:Cryptochromes (CRYs) is known as the key blue light receptors that promote photomorphogenesis in Arabidopsis, but to date, the underlying mechanisms are still not fully understood. Through interrogating the CRY2 interactome, we identified MOS4-ASSOCIATED COMPLEX subunits 3A and 3B (MAC3A and MAC3B) as blue light-independent CRY2 interacting partners. MAC3A/B proteins could be assembled into liquid nuclear condensates of CRYs in a blue light-dependent manner. Hypocotyl elongation is markedly repressed in mac3ab double knock-out mutants under various light conditions, which uncovers a previously unknown role of MAC3A/B as negative regulators in plant photomorphogenesis. Our results also uncover the noncanonical activities of MAC3A as the DNA-binding proteins that regulate transcription. Genome-wide mapping of MAC3A-binding sites reveals that blue light facilitates the binding of MAC3A to its targets, which is weakened in cry1cry2 mutants, suggesting that CRYs may enhance MAC3A activities in blue light to negatively influence photomorphogenesis. Interestingly, we observe that the genomic binding sites of MAC3A and HY5 are largely overlapped, and physical interactions between MAC3A and HY5 are detected as well. In addition, the in vitro DNA-binding assay shows that both proteins compete for the same DNA probe. These results indicate that MAC3A may antagonize the function of HY5 by competing for the common binding sites across the genome. Taken together, we propose that cryptochromes may fine-tune Arabidopsis photomorphogenesis by balancing the positive and negative effects on HY5 activities.
Project description:LONG HYPOCOTYL 5 (HY5) is a basic leucine zipper transcription factor (TF) that functions downstream of multiple families of photoreceptors. Mutations in the HY5 gene cause a myriad of aberrant phenotypes in Arabidopsis, including elongated hypocotyl, reduced accumulation of pigments, halted chloroplast development in greening hypocotyls, altered root morphology and defective hormonal and stimulus responses. HY5 thus acts as an integrater that links various gene networks to coordinate plant development. Here we report an effort to experimentally map the HY5-mediated gene networks in Arabidopsis by integrating genomic loci occupied by HY5 and HY5-dependent gene expression profiles. Our results indicate HY5 binds to over 9,000 genes, which detectably impact the expression of over 1,100 genes, either positively or negatively. Further, HY5 indirectly regulates many other genes through sub-networks mediated by other regulators. In particular, we show that HY5 regulates eight microRNA (miRNA) genes, which in turn control transcript abundance of specific target genes. Over-expressing the HY5-targeted miR408 resulted in phenotypes that are opposite to hy5 mutants. Together our results revealed both the transcriptional and post-transcriptional components of the HY5-mediated gene networks responsible for the phenotypic complexity in plants.
Project description:LONG HYPOCOTYL 5 (HY5) is a basic leucine zipper transcription factor (TF) that functions downstream of multiple families of photoreceptors. Mutations in the HY5 gene cause a myriad of aberrant phenotypes in Arabidopsis, including elongated hypocotyl, reduced accumulation of pigments, halted chloroplast development in greening hypocotyls, altered root morphology and defective hormonal and stimulus responses. HY5 thus acts as an integrater that links various gene networks to coordinate plant development. Here we report an effort to experimentally map the HY5-mediated gene networks in Arabidopsis by integrating genomic loci occupied by HY5 and HY5-dependent gene expression profiles. Our results indicate HY5 binds to over 9,000 genes, which detectably impact the expression of over 1,100 genes, either positively or negatively. Further, HY5 indirectly regulates many other genes through sub-networks mediated by other regulators. In particular, we show that HY5 regulates eight microRNA (miRNA) genes, which in turn control transcript abundance of specific target genes. Over-expressing the HY5-targeted miR408 resulted in phenotypes that are opposite to hy5 mutants. Together our results revealed both the transcriptional and post-transcriptional components of the HY5-mediated gene networks responsible for the phenotypic complexity in plants.